Development of a novel multi‑epitope vaccine against the pathogenic human polyomavirus V6/7 using reverse vaccinology

Background Human polyomaviruses contribute to human oncogenesis through persistent infections, but currently there is no effective preventive measure against the malignancies caused by this virus. Therefore, the development of a safe and effective vaccine against HPyV is of high priority. Methods First, the proteomes of 2 polyomavirus species (HPyV6 and HPyV7) were downloaded from the NCBI database for the selection of the target proteins. The epitope identification process focused on selecting proteins that were crucial, associated with virulence, present on the surface, antigenic, non-toxic, and non-homologous with the human proteome. Then, the immunoinformatic methods were used to identify cytotoxic T-lymphocyte (CTL), helper T-lymphocyte (HTL), and B-cell epitopes from the target antigens, which could be used to create epitope-based vaccine. The physicochemical features of the designed vaccine were predicted through various online servers. The binding pattern and stability between the vaccine candidate and Toll-like receptors were analyzed through molecular docking and molecular dynamics (MD) simulation, while the immunogenicity of the designed vaccines was assessed using immune simulation. Results Online tools were utilized to forecast the most optimal epitope from the immunogenic targets, including LTAg, VP1, and VP1 antigens of HPyV6 and HPyV7. A multi-epitope vaccine was developed by combining 10 CTL, 7 HTL, and 6 LBL epitopes with suitable linkers and adjuvant. The vaccine displayed 98.35% of the world's population coverage. The 3D model of the vaccine structure revealed that the majority of residues (87.7%) were located in favored regions of the Ramachandran plot. The evaluation of molecular docking and MD simulation revealed that the constructed vaccine exhibits a strong binding (-1414.0 kcal/mol) towards the host's TLR4. Moreover, the vaccine-TLR complexes remained stable throughout the dynamic conditions present in the natural environment. The immune simulation results demonstrated that the vaccine design had the capacity to elicit robust immune responses in the host. Conclusion The multi-parametric analysis revealed that the designed vaccine is capable of inducing sustained immunity against the selected polyomaviruses, although further in-vivo investigations are needed to verify its effectiveness. Supplementary Information The online version contains supplementary material available at 10.1186/s12879-024-09046-0.


Background
Polyomaviruses are small, nonenveloped DNA viruses, which are widespread in nature.Non-enveloped polyomaviruses are capable of infecting mammals and birds with their small circular double-stranded DNA genomes of approximately 5.0 kbp [1,2].Two major regulatory proteins are encoded by PyVs, the large tumor antigen (LT-ag) and the small tumor antigen (sT-ag), as well as several structural proteins (VP1 and VP2) [3].Heterologous animal models indicate that PyVs may carry strong oncogenes, that contribute to cancer in humans.Regulatory proteins are important in viral replication and transcription early in the infection cycle, while structural proteins participate in capsid formation later [4].Among 12 identified human polyomaviruses (HPyVs), six strains are involved in human diseases, especially in different human cancers.HPyV6, HPyV7, Merkel cell polyomavirus (MCPyV), trichodysplasiaspinulosa virus (TSPyV), HPyV9, MWPyV and BK virus (BKV) and JC virus (JCV), as well as newly identified viruses such as KI (KIPyV), WU (WUPyV), are the most commonly identified strains of human polyomavirus (HPyV) [5][6][7].
The World Health Organization reported increased incidence of skin cancer in the past few decades, with about 8,500 new cases of skin cancer reported daily in the United States.The relationship between HPyV6 and HPyV7 in human skin cancer has recently been elucidated, in recent decade [7,8].Several studies have been conducted to confirm the presence of HPyV6-and HPyV7-DNA in cutaneous (Table 1) and non-cutaneous (Table 2) malignancies, including malignant melanoma (MM) [9], non-melanoma skin cancer tissues, basal cell carcinoma (BCC) and squamous cell carcinoma (SCC).Studies on keratoacanthoma and trichoblastoma revealed the presence of HPyV6 in tumors [10].HPyV7-DNA was found to be detected more frequently in non-cutaneous cancers compared to HPyV6-DNA, whereas HPyV6-DNA was more commonly observed in skin malignancies.A study concluded that all age groups and genders are infected with HPyV6 and 7, and 52-93% of humans are seropositive for HPyV6 while 33-84% are seropositive for HPyV7 [7].PCR investigations demonstrated HPyV6 and HPyV7 DNA from the skin of both healthy individuals and those experiencing different types of skin tumors.Small and large T antigens are encoded by polyomaviruses, making them potentially oncogenic [11].Severe cases of HPyV6 and 7 infections are associated with skin disorders, characterized by a significant increase in viral load, expression of dyskeratotic keratinocytes, and the presence of encapsidated virions observed through electron microscopy and sequencing [12].Moreover, HPyV6 was identified in various forms of epithelial neoplasms, while HPyV7 was observed in thymic epithelial tumors.These discoveries imply that HPyV6 and HPyV7 could be crucial contributors to the development of inflammatory skin conditions and may also possess oncogenic properties [13].These findings indicate that HPyV6 and HPyV7 may have a crucial involvement in the development of inflammatory skin conditions and potentially possess oncogenic properties [13].
It has shown that HPyV6 and 7 bind, and inactivate p53 resulting in tumor progression.P53 suppresses tumor growth by regulating gene expression in response to stressors, such as DNA damage, leading to apoptosis and cell cycle arrest [20].The transactivation domain of P53 is repressed by interaction with LT-antigen preventing its binding to DNA, eventually leading to cancer in humans [21,22].
The lack of vaccine against HPyVs might be related to the complexity of the virus and the ability of HPyVs to evade the host immune system by various strategies, such as downregulating the expression of major histocompatibility complex (MHC) molecules, interfering with interferon signaling, and modulating the activity of immune cells [23].Multi-epitope vaccines contain multiple antigenic fragments (epitopes) from different proteins of the target pathogen.This type of vaccine has several advantages over traditional vaccines, such as inducing a broader and more robust immune response, reducing the risk of antigenic escape and cross-reactivity, and facilitating the production and delivery of the vaccine.Hence, a multi-epitope vaccine against HPyVs could be beneficial for preventing or treating HPyVassociated diseases, especially in immunocompromised patients who are more susceptible to viral reactivation and complications [24].
In recent years, cancer vaccines have shown promising results against different cancers but vaccine development using traditional approaches is complex and requires a significant amount of effort [25].Compared to traditional laboratory approaches, immunoinformatics enables rapid development of a multi-epitope vaccine, increasing efficiency and reducing costs [26,27].Epitope-based peptide vaccines demonstrated effectiveness in providing protective immunity against various viruses including Zika, dengue, SARS-CoV-2, and Coxsackie B viruses [28].Hence, it is supposed that a peptide-based vaccine against oncogenic polyomavirus could provide an efficient protective vaccine against HPyV6/V7 oncogenic virus strains.
To develop a multi-epitope vaccine, CTL, HTL, and LBL epitopes of the HPyV6 and HPyV7 oncoproteins including large T antigen (LTAg), VP1, and VP2 were identified and the vaccine's stability and effectiveness were analyzed by immunoinformatics methods.The study yielded compelling evidence supporting the likelihood that the multi-epitope vaccine can effectively initiate a strong anti-HyPV immune response.

Identification and evaluation of Cytotoxic T-Lymphocyte (CTL) epitopes
The important role of cytotoxic T lymphocytes (CTLs) in the host defense mechanism is known [35].CTLs have a receptor called CD8, which attaches to a molecule called MHC class I on the surface of infected cells.This enables them to deliver molecules that destroy the infection [36].The NetCTL v1.2 server(https:// servi ces.healt htech.dtu.dk/ servi ces/ NetCTL-1.2/)using weight matrix and artificial neural networks is highly efficient for epitopes prediction of 9-mer CTLs against 12 supertypes including A1, A2, A3, A24, A26, B7, B8, B27, B39, B44, B58, and B62.Using a threshold value of 0.90 to maintain a specificity and sensitivity of 0.98 and 0.74 respectively, this server was used to predict CT epitopes with high combination scores among the obtained protein sequences [37].The MHC-I binding tool of the IEDB resource was utilized to determine MHC-I binding alleles for each CTL epitope dependent on the CONSENSUS method (http:// tools.iedb.org/ mhci/) [38].To characterize antigenicity for individual CTL epitopes of the VaxiJen v2.0 server (http:// www.ddg-pharm fac.net/ vaxij en/ VaxiJ en/ VaxiJ en.html) [31], the allergenic profile of the AllerTOP v2.0 server (https:// www.ddg-pharm fac.net/ Aller TOP/) [39], toxicity prediction of ToxinPred server (http:// crdd.osdd.net/ ragha va/ toxin pred/) [40], and immunogenicity of IEDB Class I Immunogenicity tool (http:// tools.iedb.org/ immun ogeni city/) [41] were used respectively.To separate allergens from non-allergens with a prediction accuracy of 85.3% in five-fold cross-validation, the Aller-Top v2.0 server was used, which applies the amino acid descriptors, ACC transformation methods, and k-nearest neighbor (kNN) methods [39].The ToxinPred server is used for evaluation properties of different peptides using support-vector machines (SVM), that is a machine learning approach combined with a quantitative matrix for toxicity prediction [40].To confirm whether a specific epitope elicits an immune response or not, immunogenicity prediction was performed.CTL epitopes were utilized for the vaccine construction that were antigenic, non-allergenic, non-toxic, and immunogenic and showed high C-scores.

Evaluation of the human homology and epitope conservancy
We used the "epitope conservancy analysis" server (http:// tools.iedb.org/ conse rvancy/) of the IEDB resource to analyze the conservation of selected MHC class I/II epitopes.This feature demonstrates the availability of the epitope in a range of various strains.In the conservancy analysis, epitopes with 100% maximum identity were selected for vaccine construct [50].Epitope homology with the human proteome was investigated to avoid cross reaction with human proteins or weak response due to tolerance, and non-homologous epitopes were selected.Human homology was determined using the protein BLAST module of the BLAST server (https:// blast.ncbi.nlm.nih.gov/ Blast.cgi) with Homo sapiens (taxid: 9606), and a threshold of 0.05.Non-homologous peptides where no hits were found below the threshold e-value were designated as epitopes [51,52].

Population coverage analysis
Variations in the HLA allele distribution and expression in regions and races around the world may affect the response to vaccines based on epitope [60].The population coverage of the candidate vaccine was estimated by implementing the IEDB population coverage server (http:// tools.iedb.org/ popul ation/).To do this, the investigation of selected HTL and CTL epitopes coupled with their relevant HLA binding alleles in both MHC (I and II) classes were performed individually and in combination [61].In this study, our emphasis was on the global coverage of alleles and parts of different continents.

MHC cluster analysis
The MHC gene family, as one of the most polymorphic genes in the various species' genomes, contains several thousand alleles in humans [62].Cluster analysis of MHC alleles is used to identify two classes of MHC molecules with similar binding specificities.The MHCcluster 2.0 online tool (https:// servi ces.healt htech.dtu.dk/ servi ces/ MHCcl uster-2.0/) was utilized to provide highly instinctive heat maps and phylogenetic tree-based visualizations of the functional cluster between MHC variants based on the default parameters.During the MHC class I cluster analysis, the NetMHCpan-2.8approach was utilized with an HLA-prevalent and -characterized module, while for the MHC class II cluster analysis, the relevant DRB allele modules were chosen [62,63].

Designing and formulation of the multi-epitope vaccine
The vaccine was constructed from the selected HTL, CTL, and LBL epitopes of HPyV6 and HPyV7 proteins.Also, an adjuvant was attached to the vaccine structure using a suitable linker [64,65].We used TLR4 agonist as an adjuvant because viral glycoproteins were found to recognize TLR4 agonist [66,67].Therefore, 50S ribosomal protein L7/L12 was included as adjuvant (NCBI ID: P9WHE3) and attached to the N-terminal of the vaccine peptides through a bifunctional linker (EAAAK).Contrastingly, the HTL, CTL, and LBL epitopes were connected using Gly-Pro-Gly-Pro-Gly (GPGPG), Ala-Ala-Tyr (AAY), and Lys-Lys (KK) linkers, respectively [64,65].The GPGPG linker inhibits the formation of the "junctional epitope" and aids in immune processing.The AAY linker improves epitope immunogenicity by affecting peptide stability.The KK linker improves the maintenance of independent immunogenic functions of the constructed vaccine [68,69].

BLAST homology assessment
The PSI-BLAST algorithm of the NCBI Protein BLAST (BLASTp) module was used to determine the homology between the vaccine construct and the human proteome [76,77].Cross-checking study was performed to avoid autoimmune reactions through molecular imitation.The BLASTp search limited the results to records from H. sapiens (taxid: 9606) only.In order to be valid, the query coverage must not show more than 40% homology to the human proteome [78].

Homology modeling and 3D structure refinement and validation
I-TASSER tool (https:// seq2f un.dcmb.med.umich.edu//I-TASSER/) was used to generate the three-dimensional model of the multi-epitope vaccine.Using threading templates as templates, this server produces a 3D structure based on the amino acids sequence, and it estimates the C-score to assess the validity of the predicted models.The C-score of a model typically falls within the range of -5 to 2, with a higher C-score indicating greater confidence [82][83][84].Tertiary structure vaccine model refinement was performed via the GalaxyRefine tool (https:// galaxy.seokl ab.org/ cgi-bin/ submit.cgi? type= REFINE).Various parameters including GDT-HA, rmsd, poor rotations, Molprobity, clash score, and Rama-favored are produced in the output result of five refined models [85].After validation of the models through the ProSAweb online tool (https:// prosa.servi ces.came.sbg.ac.at/ prosa.php), the estimation of the Z-score and analysis of the stereochemical quality of each protein structure were performed by assessing the residue by residue geometry and overall structure geometry [86].In order to further analyze the Ramachandran plot, the Procheck web server (https:// saves.mbi.ucla.edu/) was used to determine the overall quality of the refined 3D structure of the vaccine.The Ramachandran plot is a plot of the dihedral angles phi (φ) and psi (ψ) of amino acids to visualize the percentage of amino acids in the generously allowed, disallowed, most favorite, and additional allowed areas [87].

Identification of discontinuous B-Cell epitopes
The ElliPro server (http:// tools.iedb.org/ ellip ro/) was used to estimate conformational B-cell epitopes in the designed vaccine utilizing default parameters (minimum score: 0.5; maximum distance: 6 Å).The improved Thornton's technique using residue clustering algorithms is the basis of the results.Prediction is done based on the neighbor residue clustering, protein form, and residual protein index (PI) [88].

Molecular docking of the immune receptor (TLR4) and designed vaccine
The protein data bank (RCSB) at 2.4 Å resolution was used to retrieve TLR4 complexes (PDB ID: 4G8A) [55].Heteroatoms and three chains B, C, and D were deleted in the UCSF Chimera software (version 1.11.2) [56].Energy minimization of protein was carried out using the Swiss-PDB Viewer with the GROMOS 43B1 force field [54].Molecular docking of vaccine-TLR4 complexes was performed in Cluspro (https:// clusp ro.bu.edu/ login.php) online tools [89].It's an automated, web-based program for the docking of peptide − protein or protein − protein.
The server executes a series of three computational procedures in the following manner: firstly, the process of rigid body docking is carried out employing PIPER; secondly, the 1000 docked structures with the lowest energy are subjected to clustering using pairwise IRMSD as the distance metric; and finally, the forecasted complex structures positioned at the cluster centers are refined by minimizing their energy.Also, We used the balanced coefficient to obtain the best protein-protein binding results [90].The output of this server is a short list of putative complexes ranked according to their clustering properties.

In silico immune simulation
The immune simulation study was conducted using the C-ImmSim server (https:// kraken.iac.rm.cnr.it/C-IMMSIM/ index.php? page=1) to understand and investigate the immunogenicity and immune response profile.Using the position-specific scoring matrix (PSSM), this server employs real lifelike immune responses and interactions, and machine learning [91].The time steps in the CImmSim web tool (with default parameters) were set to 1, 42, and 84, each time step is equal to 8 h and time step 1 is injection at time = 0.The time interval between two injections (a total of 3 injections) was considered 4 weeks [92].

Molecular dynamics simulation
Molecular dynamics (MD) simulation was applied to refine the TLR-vaccine complex structures using GROMACS 2018 [93,94].The structures were centered in a dodecahedron box and filled with water using tip3 water model.To neutralize systems some molecules of water were randomly replaced by Cl-or Na + .After neutralization, the energy minimization was done using steepest descent algorithm.Equilibrating the systems was performed under 100 ps NVT at temperature of 298 K followed by 100 ps NPT ensembles at pressure of 1 bar.Electrostatic interactions were calculated by PME (Flores-Canales and Kurnikova, 2015) and the LINCS procedure was applied to constrain all bonds connecting hydrogen atoms.The Final MD simulation was run for 100 ns with no restraint.
In silico cloning and codon optimization of the final vaccine protein.
The online Java Codon Compatibility Tool (JCAT) web server (http:// www.jcat.de/) was utilized for codon optimization and reverse translation of the ultimate vaccine protein [95].To express the final construct in E. coli, the K12 strain was used as the host.Using this server, important parameters such as GC content and codon adaptive index (CAI) were calculated for assessment of protein expression levels.After introducing sites for BamHI and XhoI restriction enzymes within 3′ and 5′ends of the designed vaccine sequence, respectively, this sequence was transformed into the pET30 ( +) vector through the SnapGene software.

Retrieval and evaluation of protein sequences
The reference sequence for HPyV6 and HPyV7 large T antigen (LTAg), and viral proteins 1/2 (VP1, VP2) were obtained from UniProt Proteome database.VaxiJen v2.0 tool was utilized to determine the subjected protein sequences.TMHMM v2.0 was employed to anticipate the number of TM helices.The antigenicity of candidate proteins varies from 0.4281 to 0.4996, hence the proteins of interest have sufficient predicted antigenic properties.In addition, the AllergenFP server suggested the proteins are non-allergenic.To construct a multi-epitopebased vaccine, the large T, VP1 and VP2 were included.The quantity of transmembrane helices is zero.Table 3 displays the sequences of these proteins, their UniProt entries, and allergenicity, antigenicity, and TM helices.

Identification and validation of CTL epitopes
Based on the specified selection range, 52 potential CTL epitopes of the candidate proteins were identified as nonallergenic, non-toxic, antigenic, as well as immunogenic (Table S1).The epitopes were calculated with NetCTL 1.2 using a combinatorial approach.Just ten predicted CTL epitopes were selected for peptide-based vaccine design.The list of the chosen CTL epitopes with their characteristics in the final vaccine is shown in Table 4.

Identification and validation of HTL epitopes
Overall, 55 potential HTL epitopes under specified selection range as non-allergenic, non-toxic, and antigenic were identified (Table S2).Evaluation of expected cytokine induction capability was done on selected HTL epitopes and based on those results 7 peptides were selected to include in the final vaccine.The list of the chosen HTL peptides with their characteristics in the final vaccine is shown in Table 5.

Identification and validation of linear B-cell epitope (LBL)
Overall, 46 LBL epitopes from the target proteins were identified by evaluating potential toxicity, immunogenicity and antigenic characteristics (Table S3).One LBL epitope was chosen from each of the 6 protein components for usage in the ultimate vaccine.The list of the six LBL epitopes with their characteristics in the final vaccine is shown in the Table 6.

Vaccine evaluation Evaluation of human homology and epitope conservancy
A lack of homology to normal human proteins was assessed for each of the shortlisted epitopes in both MHC classes and no homologies were identified in the human proteome, suggesting that responses against these peptides are not likely to cause response against a normal protein.Tables 4 and 5 have incorporated conservancy and human homology analyses of selected epitopes.

MHC cluster analysis
MHC cluster v2.0 server was exploited in order to cluster MHC classes I and II alleles that interact by selected structural protein epitopes.In this study, 25 alleles from the MHC class I, and 22 alleles from the MHC class II were analyzed.MHCI and MHC II molecules Cluster analysis is shown in Fig. 3A, C, respectively.A tree map showing the cluster analysis of MHCI and MHCII is also shown in Fig. 3B, D. The red zones on the heat map were associated with stronger interactions, whereas the yellow zones were associated with feeble interactions among clusters of both MHC molecules.

Formulation of the vaccine construct
In order to formulate the vaccine construct, we assembled the most favorable CTL, HTL, and LBL epitopes using AAY, GPGPG, and KK linkers, respectively.Furthermore, 50S ribosomal protein L7/L12 adjuvant (NCBI ID: P9WHE3) was attached using EAAAK linker to the N-terminal region of the vaccine.The structure of the vaccine consists of seven epitopes of HTL, ten epitopes of CTL, and six epitopes of LBL from the target protein sequences of polyomaviruses 6 and 7.After suitable evaluation and comparison of different structures, we determined the final structure of the vaccine with 501 amino acids.The final recombinant vaccine was analyzed for subsequent evaluations (Fig. 4).

Vaccine assessment Assessment of the antigenicity, allergenicity, and physicochemical properties of the final vaccine protein
The physicochemical characteristics of the formulated construct were evaluated.The vaccine's chemical formula is C 2455 H 3814 N 662 O 694 S 11 .The molecular weight of the vaccine construct was estimated to be 54059.97Da.It was calculated that the protein has a theoretical pI value of 9.41.This value represents that the protein is highly basic.The grand average of hydropathicity (GRAVY) property demonstrated the hydrophilic nature of protein, as it was-0.317.Furthermore, the instability index We also employed several tools to evaluate the solubility of the vaccine sequence.In Solpro and Protein-Sol tools, the score of solubility was estimated to be 0.777639 and 0.504, respectively.Also, the final proposed vaccine did not indicate any signal peptides and Transmembrane helices based on the prediction data (Table 9 and Fig. 5A).

BLAST homology assessment
Homo sapiens proteins were found to be 24% homologous with the protein vaccine sequence based on sequence homology between the designed vaccine and the proteome sequence.These results confirmed that chimeric vaccine construct cannot prompt autoimmune responses in the host based on BLAST homology assessment.This study focused on the Homo sapiens species (taxid:9606).

Tertiary structure modeling, refinement, and validation of the multi-epitope vaccine
The I-TASSER online tool was utilized to make the tertiary structure of the ultimate vaccine protein.
The server created 5 models for the appointed vaccine.The estimated C-score values for models 1-5 were -0.97, -1.63, -2.97, -4.44, and -3.40, respectively.The best structure with a C-score value of − 0.97 from modeling was selected for additional analysis.before refinement, Procheck and ProSA and tools were utilized to assessment this model.In the current survey, model 1 showed a z-score of -2.12 and 73.4% of the residues in the most favored regions.The GalaxyRefine tool was used to refine the 3D structure of the submitted model.This generated five refined structures for the raw model (Table 10).After refinement, all structures show the regions favored by Rama more than the submitted originally raw model.Model 3 was determined to be the best refined structure among the generated models.It displayed goodRama-favored (90.6), poor rotamers (0.8), MolProbity (2.195), clash score (14.4), rmsd (0.517), and GDT-HA (0.9037) scores.ProSA and SAVES v6.0 online tools were employed to validate the refined structure.Based on Ramachandran plot of the selected model indicated that 87.7% of amino acids in favored regions, 9.6% additional allowed, 1.0% generously allowed, and 1.7% disallowed regions were found.The Z-score value for the refined model was estimated -2.61 (Fig. 6).For further analysis, we have selected model 3 in this study.

Screening for conformational B-Cell epitopes
The ElliPro server identified thirteen Conformational B-cell epitopes in the vaccine construct sequence (Fig. 7).A total of 243 residues were found in these epitopes  ranging in size from 3 to 98, shown in Table 11.Moreover, B-cell epitope scores ranged from 0.975 to 0.53 for the prediction of conformational B-cell epitopes.

Molecular docking of the vaccine protein and TLR complex
Immune cells and vaccine constructs must interact in order to produce a stable and efficient immune response.Molecular Docking of the designed vaccine with TLR4 was carried out by the ClusPro 2.0 server.
In the current study, the program produced 30various clusters and ranked them by energy level.There were -1414.0,-1406.2,-1372.0,-1350.9,-1341.0,-1339.5, -1327.2, and -1321.0kcal/mol of energy in the eight top clusters.The best group with the minimum energy of -1414.0kcal/mol was selected.The Chimera 1.15rc program was applied to visualize the docked complex (Fig. 8).Using the LigPlot v1.4.5 software, we have generated a map with the hydrophobic interactions and hydrogen bonds between the protein vaccine and TLR4 (Fig. 9).The vaccine and chain A of TLR4 formed 20  hydrogen bonds.These hydrogen bonds are formed by amino acids along with their lengths, as shown in Table 12.

Vaccine immune simulation In silico immune simulation
Immune simulator C-ImmSim was employed to provide simulations of the immune responses associated with the final chimeric vaccine construct.The secondary and tertiary responses were clearly indicated by the anticipated elevated levels of IgM + IgG, IgM, IgG1 + IgG2, and IgG1 antibodies, subsequently followed by a reduction in antigen concentration (Fig. 10A).Results indicated a variety of long-lasting B-cell isotopes.B-cell isotype switching and memory formation may be involved in this process (Fig. 10B).In addition, T helper (helper) and TC (cytotoxic) cells are showing a clear increase with memory growth (Fig. 10C and D).There is also a clear increase in IFN-γ production and the growth of dendritic cells after immunization (Fig. 10E and F).These data represent that After successive exposures to the target antigen, robust and significant secondary immune response, antigen clearance enhancement, and production of vigorous immune memory occur.

Evaluation of MD simulations
The global structural stability of proteins was evaluated using Root Mean Square deviation (RMSD) of the backbone atoms.This plot shows how much the protein conformation has changed during MD simulation from initial structure.The TLR showed RMSD value in the range of 0.25 to 0.35 nm.The RMSD after 20 ns reached stability.Furthermore, the RMSD of vaccine was plotted and was in the range of 0.45 to 1.The root-mean-square fluctuation (RMSF) indicates the fluctuation of protein residues over time from a reference position during simulation.In current simulations, no unusual fluctuation

Codon optimization and in silico cloning of the designed vaccine
Codon optimization is used to ameliorate gene expression and translation precision of the recombinant protein by adapting to the target host's codon bias.Reverse translation of the predicted vaccine was performed to achieve maximum expression in Escherichia coli strain K12 by Jcat server.After codon optimization, CAI score and GC content in improved protein sequence were estimated at 1.0 and 52.96, respectively.The data demonstrate that the improved protein sequence could be expressed sustainably in the E. coli system.Finally, the improved sequence was successfully integrated into the pET30a ( +) vector by SnapGene program (Fig. 12).

Discussion
Conventional techniques for vaccine development involve the use of whole organisms, which can lead to undesired exposure to antigens and may trigger allergic responses.To prevent allergic responses, peptidebased vaccines that included short peptide fragments derived from immunogenic proteins have been used to produce strong and targeted immune reactions.Rabies, rubella, yellow fever, smallpox, hepatitis A/B, chickenpox, polio, influenza, Human Papillomavirus, and Japanese encephalitis are some of the infectious diseases which vaccines are highly effective against [96,97].The development of vaccines involves complex, timeconsuming, and expensive in vitro and in vivo assays to  ensure vaccine effectiveness [98].Current advances in immunoinformatics and computational biology allow the design of effective vaccines in silico and reduce the number of in vitro experiments [99].Using an in vitro study, an experimentally validated multiepitope vaccine was designed against Clostridium perfringens [100].With this method, a wide range of vaccine candidates can be identified without the requirement of cultivating pathogenic organisms [98].Human polyomavirus 6 (HPyV6) and HPyV7 are polyomaviruses species initially discovered in the skin of healthy people [101].The role of HPyV6 and 7 proteins in binding and inactivating p53 has been documented, suggesting its oncogenic role [102].The incidence of malignant skin tumors has increased over recent decades, chiefly as a result of alterable exposures.The World Health Organization reported that about 8500 new cases of skin tumors are diagnosed every day in the U.S. [7,8].Several studies have shown the prevalence of HPyV6-and 7 in primary cutaneous malignancies, including actinic keratosis, basal cell carcinoma, bone marrow transplantation, neuroendocrine, and lymphoid skin cancers [7].
Therefore, in the present study, the POLY capsid protein VP1, POLY minor capsid protein VP2, and POLY large T antigen from HPyV6 and HPyV7 were examined as candidate antigens for epitope identification.The allergenicity, toxicity, and antigenicity of the identified epitopes were assessed.There are a number of factors to consider when making peptide-based vaccines, including the intrinsic properties of the selected epitopes, adjuvant, and linker, and their arrangement and location within the protein.Based on the findings from the studies conducted by Olugbenga et al. [103], Mahnoor Majid et al. [104], and Sami et al. [99], we used KK, GPGPG, and AAY linkers to fuse LBL, HTL, and CTL epitopes, respectively.Epitope presentation is promoted with AAY and GPGPG linkers, while junctional epitopes are reduced with these linkers [105,106].The KK linker, a bi-lysine basic linker, preserved the immunogenic properties of B cell epitopes while keeping the pH near physiological levels [107,108].
Compared with live attenuated vaccines, computational vaccines have relatively low immunogenicity.In order to address this problem, adjuvants are routinely employed.Hence, adjuvants have been widely used to increase vaccine effectiveness.Adjuvants generally function by activating innate immune cells through pathogen associated molecular pattern receptors.Adjuvants can also improve vaccines by stabilizing the epitope structure of the vaccine antigen, creating a suitable source for the gradual release of the antigen, better presenting the antigen to the antigen-presenting cells (APC), increasing the absorbing molecules of these cells at the site of the vaccine, and the proper binding of the antigen to these cells improves the vaccine performance [109].The 50S ribosomal protein L7/L12 (Locus RL7_MYCTU) from Mycobacterium tuberculosis is a TLR4 agonist [110].Thus, in order to enhance the immunogenicity of vaccine, we used it as an adjuvant.EAAAK, an empirical α-helical linker, reduces the connection with other protein regions while providing rigidity and improving chimeric protein durability [111].Multiple servers determined that the construct vaccine was non-allergenic and highly antigenic, demonstrating that triggers robust immune responses without inducing unwanted allergies.
The final vaccine had a theoretical pI of 8.3, indicating its alkaline nature.Furthermore, the vaccine construct exhibited an average molecular weight of 54.05 KDa, indicating its favorable antigenic characteristics [112].According to standards, proteins with a molecular weight below 110 kDa are deemed appropriate vaccine candidates [113].The instability index of the vaccine was measured as 34.63,Values less than 40 are considered as a stable protein in biological environments [114].The constructed vaccine has an indicated average half-life of above 20, 10, and 30 h in yeast cells (in vivo), E. coli (in vivo), and mammalian reticulocytes (in vitro), respectively.On the basis of previous findings, these half-life results are acceptable [99,115].The aliphatic index was 72.95, indicating the constructed vaccine would be thermostable at natural human body temperature [116].The GRAVY value of the protein was -0.317, indicating the hydrophilic nature of the vaccine.Vaccine formulation and purification are made easier by the strong affinity for water molecules [117,118].
Fig. 12 The map of the in silico cloning of the vaccine construct into the pET30a ( +) vector using SnapGene software.The black segment indicates the backbone of the vector and the red segment shows the vaccine construct.This vaccine construct contains restriction sites for XhoI and BamHI restriction enzymes at the 5′ and 3′ ends, respectively After making the 3D model of the vaccine, the refinement system is employed to enhance its quality, both in terms of global and local structures.Validation of the model is necessary to accurately compare the unrefined structure with the refined structure.The Ramachandran plot indicated that 73.4% of the amino acid residues in the unrefined structure were detected in the desired region, while 87.7% of the amino acid residues in the refined structure were placed in the desired region, demonstrating improvement in the refined structure.Assessment of the immune response induced by an antigen is one of the primary characteristics in the validation of an introduced vaccine [119].Molecular analyzes were employed to investigate the molecular connection between the formulated vaccine and TLR4, and suitable interactions were detected with a strong affinity score of -1414.0kcal/mol.This relationship of the engineered vaccine with TLR-4 demonstrated that the recombinant protein vaccine has the capacity to stimulate an innate and adaptive immune response.To investigate the stability and dynamic efficiency of the vaccine/TLR4 complex, MD simulation was performed and the RMSD diagram confirmed the stable binding of this compound.
An appropriate host is required for the expression of recombinant protein.E. coli expression systems is the most common host for expressing recombinant proteins [120,121].To enable the recombinant vaccine to be expressed at high levels in E. coli (K12 strain) codon optimization was performed.An analysis of the designed vaccine indicated a CAI score of 1.0 and GC content of 52.96.CAI values of more than 0.8 and GC content of 30-70% have been reported to favor high expression in the E. coli host [122,123].

Conclusion
Human polyomaviruses (HPyVs) infect a wide range of tissues such as skin, kidney and respiratory tract and often lead to persistent and asymptomatic infection, while these infections can lead to cancer.Currently, no significant therapeutic vaccine is available for HPyV.In this study, immunoinformatics techniques were applied to identify and refine candidate vaccines against HPyV.The highly immunogenic T and B cell epitopes were identified and used for vaccine design.The proposed vaccine is projected to produce robust immune reactions, including cytokines, and interferons.The binding analysis confirmed the vaccine binding to the immune receptor TLR4 that was dynamically stable.Although experimental trials in appropriate animal models is necessary to test the potency of the engineered vaccine, analysis using different bioinformatics tools indicated the high immunogenicity and preventive potential of the developed vaccine.

Fig. 1
Fig. 1 Molecular docking of the selected CTL epitopes with their respective HLA alleles as indicated in Table 5

Fig. 3
Fig. 3 Results of the Cluster analysis for MHC I and II molecules.A Heat map showing the MHC-I cluster, B Heat map showing the MHC-II cluster, C detailed tree map of the MHC-I clustering analysis, D detailed tree map of the MHC-II clustering analysis

Fig. 4
Fig. 4 Schematic presentation of the formulated multi-epitope vaccine construct.The multi-epitope vaccine constructs included (left to right) an adjuvant and CTL, HTL, and LBL epitopes, indicated in the Brown, navy, light blue, and violet rectangular boxes, respectively

Fig. 5
Fig. 5 Protein-Sol server prediction of vaccine protein Solubility (A), Prabi server Prediction of vaccine secondary structure (B), PSIPRED server Prediction of vaccine secondary structure (C)

Fig. 8 Fig. 9
Fig. 8 Three-dimensional representation of molecular docking of the vaccine construct and TLR complex

Fig. 10
Fig. 10 Results of the in silico immune simulation using the C-ImmSim server for the designed vaccine.A the generation of immune complex and immunoglobulin as a result of response to designed vaccine injections, B B lymphocyte total count after the three injections, C growth of CD4 T-helper lymphocytes after the three injections including active, duplicating, resting, anergic, D Increasing the number of cytotoxic CD8 lymphocytes after injection of the designed vaccine, E Proliferation of dendritic cells after immunization, F Stimulation of cytokines and interleukins after vaccine administration

Fig. 11
Fig. 11 Molecular dynamics simulation of the TLR4 complex and vaccine construct.A RMSD plot of the vaccine construct, B RMSD plot of the TLR4, C RMSF plot of the vaccine, D RMSF plot of the TLR4, E and F radius of gyration of the vaccine-TLR4 complex, and G hydrogen bond analysis from the simulation system

Table 3
Details of the selected proteins and Their Selection Criteria

Table 4
A brief list of CTL Epitopes to form the final structure of the vaccine

Table 6
A brief list of LBL Epitopes to form the final structure of the vaccine

Table 7
Data of the molecular docking between CTL Epitopes and HLA Alleles Fig. 2 Analysis of population coverage of alleles worldwide

Table 8
Analysis of MHC restriction data for worldwide population coverage a Projected population coverage b Average number of epitope hits/HLA combinations recognized by the population c Minimum number of epitope hits/HLA combinations recognized by 90% of the population Population/

Table 9
Allergenicity, antigenicity, and physicochemical properties of the final structure of the vaccine

Table 10
Models of vaccines refined by Galaxy Refine

Table 11
Shortlist of Conformational Epitopes of the ultimate designed vaccine

Table 12
Hydrogen bonding interactions between TLR4 and vaccine amino acids